Bismuth(III) compounds and methods thereof

ABSTRACT

The present disclosure relates to a pharmaceutical composition comprising: (a) β-lactam antibiotics and (b) a metallo-β-lactamases (MBLs) inhibitor. The inhibitor relates to Bi(III) compounds or the pharmaceutically acceptable salts thereof. The present patent also provides methods of making Bi(III) compounds or the pharmaceutically acceptable salts thereof. Also provided is a method for treating MBLs-producing bacterial infection using a metal replacement mechanism.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application under 35 U.S.C. 111(a) andclaims priority to of U.S. Ser. No. 15/278,916, filed Sep. 28, 2016, nowU.S. Pat. No. 10,201,518, which is incorporated by reference in itsentirety.

1. INTRODUCTION

Disclosed herein is a pharmaceutical composition comprising: (a)β-lactam antibiotics and (b) a metallo-β-lactamases (MBLs) inhibitor. Inone embodiment, the inhibitor relates to Bi(III) compounds or thepharmaceutically acceptable salts thereof. More specifically, thepharmaceutical composition comprises effective amounts of: (a) aβ-lactam antibiotic; and (b) Bi(III) compounds or the pharmaceuticallyacceptable salts thereof. Also provided is a method for preventing ortreating MBLs-producing bacterial infection. The present disclosure alsoprovides methods of making the composition that comprises the β-lactamantibiotic and the MBLs inhibitor. The disclosure also relates to themodulation of MBL activity by Bi(III) compounds. The MBLs inhibitorinhibits MBLs using a metal replacement mechanism. In certainembodiments, the disclosed composition comprises a compound that is abroad-spectrum anti-bacterial agent for treating topical, local and/orsystemic bacterial infections. In certain embodiments, the disclosedcompound is used in the treatment of infections caused by MBL-producingbacterial pathogens. Provided herein is a medical device comprising acoating comprising the compound disclosed herein. Provided herein is amethod of making a medical device comprising a coating comprising thecompound disclosed herein. Provided herein is a method of making ananti-biofilm surface.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 9, 2020, isnamed 10030/005364-US1_ST25.txt and is 1.04 KB in size.

2. BACKGROUND

Beta-lactam antibiotics are the most widely used antibacterial drugs inthe treatment of bacterial infections. As a fierce fight back, bacteriaproduce enzymes called β-lactamases, which break down the β-lactams,leading to a broad-spectrum resistance to this class of antibiotics. Theemergence and spreading of β-lactamases constitutes an enormous threatto public health globally.¹⁻³

Based on the unique enzymatic mechanisms, β-lactamases can befunctionally classified to two major types, i.e. serine-β-lactamases(SBLs) and metallo-β-lactamases (MBLs) with the former employing aserine as a nucleophile and the latter using zinc ions to breakdownβ-lactam ring.⁴ MBLs are considered as the more injurious β-lactamasesconferring a broad-spectrum of resistance to β-lactam antibiotics due totheir unique enzymatic mechanism.⁵⁻⁷ This is largely due to thefollowings: (1) resistant determinants of MBLs are often encoded onmobile genetic elements and could easily spread among/across a varietyof bacterial species by horizontal gene transfer. The predominant MBLsproducers, Enterobacteriaceae including Escherichia spp., Klebsiellaspp., Pseudomonas spp., Acinetobacter spp. and Enterococcus. spps.,could be easily found in community and even health-care context, andspread among people by hand carriage, food and water.⁸⁻¹¹ (2) MBLs havestrong β-lactamase activity and are able to hydrolyze or inactivate themost commonly used β-lactam antibiotics, such as cephalosporins andcarbapenems. Furthermore most MBLs producing enterobacterial strains areable to co-express other types of resistance genes including thoseencoding other β-lactamases (AmpC, ESBL, OXA-48 and KPC) and resistancedeterminants for other antibiotics including fluoroquinolones (qnrA6 andqnrB1), aminoglycosides (armA, rmtA and rmtC), acrolides (ereC),rifampicin (arr-2) and sulfonamide (sul-2).^(4,12,13) The latest exampleof MBLs is New Delhi metallo-(3 lactamase-1 (“NDM-1”). In 2009, aSwedish patient was firstly reported to be infected by NDM-1 producingKlebsiella pneumoniae with resistance to multiple antibiotics includingall carbapenems upon the return from India.⁹ Ever since, NDM-1 has beenspread to all inhabited continents wherein Indian subcontinent and Chinaemerge as the two biggest reservoirs, and therefore NDM-s is oftenregarded as the notorious “superbug” in mass medium.¹⁴⁻²³ However, thereappears no effective treatment for the infection caused by NDM-1.

There is a lack of inhibitor specifically targeting MBLs availableclinically up to date. MBLs are considered more menacing than SBLs owingto the following two aspects: (1) the architectures of active sites ofMBLs vary greatly among microorganisms. Thus it remains to be a greatchallenge to design an inhibitor against all MBLs among differentbacteria. (2) Unlike SBLs, MBLs have no or few stable reactionintermediates, which immensely increase the difficulty in copying theinhibition mode of SBLs inhibitor, such as clavulanic acid.^(7,12)

Till now, considerable efforts have been made for the development of MBLinhibitors. A representative MBL inhibitor is Aspergillomarasmine A(AMA) reported in Nature, 2014, which is effective against MBLs (mainlyfor NDM-1 and VIM-2) among a variety of gram-negative bacteria andexerts good in vivo efficacy against K. pneumonia (NDM⁺).²⁸ Anotherexample is a rhodanine-derived thioenolate showing a potentbroad-spectrum activity against MBLs. The thioenolate is found to bindVIM-2 via di-zinc chelation by crystallography.²⁹ Since then, quite afew MBL inhibitors began to emerge and make progress in this area, buttheir in vivo efficacies have not been proved yet. Above instancesmirror the conventional way to deal with MBLs, i.e. designing inhibitorswhich are able to coordinate to or chelate Zn(II) in the active sites,such as carboxylic acids and thiol-containing compounds. But such astrategy usually suffer from relatively poor selectivity and lowefficacy and is unlikely to develop a wide-spectrum of MBL inhibitors,not to mention the hidden worry of the generation of resistance to thoseorganic inhibitors by microorganisms. Furthermore, though someFDA-approved drugs, such as DL-captopril, glutathione and2,3-dimercaprol,³⁰ have been used in the studies, no clinicallyavailable MBL inhibitor has been approved yet. Therefore, there appearsto be lack of developments of the relevant MBLs inhibitors.

3. SUMMARY

Provided herein is a composition comprising: (a) β-lactam antibiotics;and (b) metallo-(β-lactamases (MBLs) inhibitor. The inhibitor relates toBi(III) compounds or pharmaceutically acceptable salts thereof, thatmodulate the activity of MBLs via an unprecedented metal replacementmechanism. In addition, MBLs inhibitors are efficient β-lactamantibiotic partners for the treatment of infection caused byMBL-producing bacterial pathogens. In certain embodiments, infectionsthat are treated by the disclosed composition are caused by bacteriathat are resistant to β-lactam antibiotics. In certain embodiments, theβ-lactam antibiotics have the following core structure:

The β-lactam core structures. (A) A penam. (B) A carbapenam. (C) Anoxapenam. (D) A penem. (E) A carbapenem. (F) A monobactam. (G) A cephem.(H) A carbacephem. (I) An oxacephem

In certain embodiments, the β-lactum antibiotics are penicillins,cephalosporins, and carbapenems. In certain embodiments, the bacteriaare gram-negative. In certain embodiments, the bacteria producemetallo-β-lactamases (“MBL”s). In certain embodiments, the MBLs areimipenemase (“IMP”), Verona integrin-encoded metallo-β-lactamase (“VIM”)and New Delhi metallo-beta-lactamase (“NDM”).

Provided herein are methods of preparing Bi(III) compounds by eitherrepositioning of Bi(III) drugs or coordinating Bi(III) to N, O, or Scontaining ligands. Examples of the ligands of Bi(III) compoundsinclude, but are not limited to,

Provided herein are composition comprising a Bi(III) compound orpharmaceutically acceptable salt thereof. In certain embodiments, theBi(III) compound or pharmaceutically acceptable salt thereof is aBi(III) complex. In certain embodiments, the Bi(III) complex includesthe complexes listed in Table 1.

Also provided are methods of using Bi(III) compounds for the modulationof MBL activity. Provided herein is a method of treating bacterialinfection. Provided herein is a pharmaceutical composition comprising:(a) a β-lactam antibiotic; and (b) Bi(III) compounds or pharmaceuticallyacceptable salts thereof, as a medicament for the treatment of theMBL-producing bacterial infection. In certain embodiments, the Bi(III)compounds are bismuth subsalicylate (“BSS”), bismuth subgallate (“BSG”),colloidal bismuth subcitrate (“CBS”) and ranitidine bismuth citrate(“RBC”).

The composition described herein also exhibited potent anti-biofilmactivity against bacterial biofilms that are resistant to currentlyavailable antibacterial agents. In certain embodiments, the compositionis an antibacterial agent against a broad spectrum of bacterialinfections. Also described herein is a composition comprising Bi(III)compounds. In certain embodiments, the composition is a pharmaceuticalcomposition that includes solutions, suspensions, gels, fluid gels,emulsions, emulsion gels, lotions, ointments, film forming solutions,creams, sprays and lacquers. In particular, the antibacterialcomposition is used to treat or prevent local or systemic bacterialinfection in a subject. In specific embodiment, the subject is a mammal.In specific embodiment, the subject is human. In one embodiment,provided herein is a method of treating bacterial infection comprisingadministering to a subject, a pharmaceutical formulation comprising atherapeutically effective amount of one or more antibacterial agents incombination with a Bi(III) compound.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the basic dimeric unit of exemplary clinically used bismuthcompounds, colloidal bismuth subcitrate (CBS).

FIG. 2 shows the cell viability test by XTT assay upon treatment ofhuman liver cells (MIHA) with different amounts of Bi(III) compounds.

FIG. 3 shows the inhibition of enzymatic activity of purified NDM-1 andVIM-2 by CBS with IC₅₀ values of 5.83 μM and 44.45 μM respectively.

FIG. 4 shows the comparison of the hydrolysis rates of meropenem (MER)by native NDM-1 and Bi-bound NDM-1 (Bi-NDM-1). The corresponding V_(max)and K_(m) values are summarized in Table 3

FIG. 5 shows the substitution of Zn(II) in NDM-1 by Bi(III) asdetermined by ICP-MS. With addition of increasing amounts of CBS to thenative NDM-1 samples, Zn(II) was gradually released and binding of onemolar equivalent of Bi(III) led to two molar equivalents of Zn(II) to bereplaced from NDM-1.

FIG. 6 shows different UV-vis spectra of apo-NDM-1 upon addition of 0.2to 1.5 molar equivalents of Bi(III). The inset shows the changes ofabsorbance at 340 nm.

FIG. 7 shows the comparison of the enzyme activities of apo-NDM-1 andBi-NDM-1 upon supplementation of various concentrations of ZnSO₄.

FIGS. 8 (a)-(c) show (a). SDS-PAGE of purified NDM-1 expressed in E.coli (BL21) cells harboring the NDM-1 gene in pET-28a vector. (b) Aphoto of the crystals grown from purified native NDM-1. (c) Diffractionimage shows reasonable diffraction of the crystal (data were collectedat BL17U1 station in Shanghai Synchrotron Radiation Facility).

FIGS. 9 (a)-(b) show (a) X-ray structure of the active site of nativeNDM-1 with two Zn(II) ions shown in grey spheres and the bridginghydroxyl nucleophile in a red sphere. (b) X-ray structure of the activesite of Bi-bound NDM-1 with the anomalous density of Bi contoured at 1σshown in purple.

FIGS. 10 (a)-(b) show a heat map of checkerboard MIC analysis over MERand CBS against clinical isolates of (a) E. coli (NDM-1⁺) and (b) E.coli (NDM-1⁻).

FIG. 11 shows the percent cell viability of E. coli (NDM-1⁺) upon theexposure to different antibiotics in the absence or presence of Bi(III)compounds. Note that evident restoration of activities of antibioticswas observed. The concentrations used in this test represent are: CBS 32g mL⁻¹, Bi-NAC 16 μg mL⁻¹, all the antibiotics 32 μg mL⁻¹. Abbreviation:AMOX: amoxicillin; AMP: ampicillin; NAF: nafcillin; CEF: cefdinir.

FIG. 12 shows a time-kill curves for log-phase growing E. coli (NDM-1⁺)upon treatment with MER, CBS and their combination for up to 24 hours.The concentrations of the drugs are 16 μg mL⁻¹ and 32 μg mL⁻¹ for MERand CBS, respectively.

FIG. 13 shows a heat map of the mutation frequency of E. coli (NDM-1+)exposed to identical concentration of either MER or combination of MERand CBS.

FIGS. 14 (a)-(b) show a bacterial density upon treatment of MER atescalating concentrations, in the absence or presence of CBS (32 μgmL⁻¹) in (a) cell-associated model and (b) cell-invaded model.

FIG. 15 is a survival curve showing efficacies in a murine peritonitisinfection model. BALB/c mice were infected by a lethal dose of E. coliclinical isolate (NDM-1) via i.p. injection and treated with one doseand 4 h post infection, followed by twice-daily treatment via i.p.injection. Five groups of mice were treated with MER (5 mg kg⁻¹), CBS(20 mg kg⁻¹), the combination of MER (5 mg kg⁻¹) and CBS (20 mg kg⁻¹),CBS (20 mg kg⁻¹, in the absence of bacteria) and PBS, respectively.P<0.001, Mantel-Cox test.

4.1 Definition

The term “antibiotics” herein refers to compounds that either kill orinhibit the growth of bacteria.

The term “metallo-β-lactamase” herein refers to a type of metallo-enzymeproduced by bacteria endowing them resistance to β-lactam antibiotics bycatalyzing the hydrolysis of the amide bond in the β-lactam rings, andthus demilitarizing their antibacterial properties.

The term “inhibitor” described herein refers to a molecule that binds toan enzyme and hinder the enzyme from its catalytic reaction.

The term “Bi(III) compounds” refers to bismuth drugs for example,including but not limited to bismuth subsalicylate (“BSS”), bismuthsubgallate (“BSG”), citrate based bismuth compounds e.g. colloidalbismuth subcitrate (“CBS”) and ranitidine bismuth citrate (“RBC”), orBi(III) compounds comprise complexes with Bi(III) coordinated to but notlimited to N, O or S containing ligands. The ligands involved include,but are not limited to,

The term “cytotoxicity” refers to the property of being toxic to cells.

The term “MBL-producing bacterial pathogens” and “MBL-producingbacteria” refer to the bacterial pathogens that produce MBL(s) naturallyor by the inducement of molecular biology reagent, such as isopropylβ-D-1-thiogalactopyranoside (IPTG).

The term “clinically relevant susceptible range” refers to the zone ofinhibition or MICs at which an organism is considered to be susceptiblebased on obtainable serum concentrations of the drug, test compounds andclinical trials.

The term “synergistic effect” refers to the interaction between two ormore compounds or chemicals when the combined effect is larger than thesum of the effects of the individual components.

The term “in vitro” refers to the experimentation carrying out withmicroorganisms, cells, and biological molecules outside their normalbiological context. The term “in vivo” refers to experimentation using awhole, living organism as opposed to a partial or dead organism, whichis most commonly represented by animal studies and clinical trials.

The term “biochemical methods” refers to those routine experimentaltechniques used in the field of biochemistry, which herein includemolecular cloning, protein expression and purification as well asprotein characterization. ‘Molecular cloning’ refers to a general methodto engineer a desired DNA fragment into a vector for holding andpreserving the fragment as well as directing their self-replicationsinside host cells for the ease of protein expression and purification.‘Protein expression’ involves the use of some small molecules tostimulate host cells to produce the desired protein from the DNAintracellularly in a large amount enough for experimental uses. ‘Proteinpurification’, refers to separate the desired proteins from otherunwanted molecules present inside cells so as to greatly enhance thehomogeneity of the protein sample obtained.

The term “protein characterization” refers to the various physical andbiochemical techniques used to elucidate the structure and function of apurified protein. The term ‘metal content’ refers to the ratio of metalion(s) to a protein in various metallo-proteins. The term ‘enzymeactivity’ refers to the molar quantity of substrate converted per unittime and is a standard parameter to evaluate and compare the reactionrate of an enzyme either in the presence or in the absence ofinhibitors.

The term “pharmaceutically acceptable salt” refers to any salt(s) of acompound provided herein which retains its biological properties andwhich is not toxic or otherwise undesirable for pharmaceutical use. Suchsalts may be derived from a variety of organic and inorganiccounter-ions well known in the art.

The term “solvate” includes a compound provided herein or a saltthereof, that further includes a stoichiometric or non-stoichiometricamount of solvent bound by non-covalent intermolecular forces.

The terms “subject” and “patient” are used interchangeably herein. Theterms “subject” and “subjects” refer to an animal, such as a mammalincluding a non-primate (e.g., a cow, pig, horse, cat, dog, rat, andmouse) and a primate (e.g., a monkey such as a cynomolgous monkey, achimpanzee and a human), and for example, a human. The term “a subjectin need thereof” refers to a subject having a bacterial infection, or asubject at risk of developing a bacterial infection. The subject mayhave been diagnosed as having such a bacterial infection as describedherein or using standard medical techniques known to those of skill inthe art. Alternatively a subject may exhibit one or more symptoms ofbacterial infection.

The terms “compound”, “agent” and “drug” are interchangeable.

The terms “therapeutic agent” and “therapeutic agents” refer to anyagent(s) which can be used in the treatment or prevention of aninfection. In certain embodiments, the term “therapeutic agent” includesa compound provided herein. In one embodiment, a therapeutic agent is anagent which is known to be useful for, or has been or is currently beingused for the treatment or prevention of the infection.

The term “therapeutically effective amount” includes an amount of acompound or composition that, when administered to a subject fortreating an infection, is sufficient to effect such treatment. A“therapeutically effective amount” can vary depending on, inter alia,the compound, the infection and its severity, and the age, weight, etc.,of the subject to be treated.

The term “treating” or “treatment” of any infection refers, in oneembodiment, to ameliorating the infection that exists in a subject. Inanother embodiment, “treating” or “treatment” includes ameliorating atleast one physical parameter, which may be indiscernible by the subject.It is intended to include preventing, ameliorating, curing, reducingbacterial growth, or preventing any increase in bacterial growth.

The term “reducing bacterial growth” includes an interference inbacterial cell growth or processing which can be determined by areduction in cell number, a reduction in cell division.

The term “about” refers to ±0.5 for a numerical value.

The term “resistant”, “resistance” and “develop resistance” when referto bacteria or bacterial infections that are no longer responsive to acompound or drug that was previously effective in reducing the bacterialgrowth or preventing any increase in bacterial growth.

5. DETAILED DESCRIPTION 5.1 Metallo-β-lactamases Inhibitors

In the following detailed description, numerous specific details are setforth to provide a thorough understanding of claimed subject matter.However, it will be understood by those skilled in the art that claimedsubject matter may be practiced without these specific details. In otherinstances, methods, apparatuses, or systems that would be known by oneof ordinary skill have not been described in detail so as not to obscureclaimed subject matter. It is to be understood that particular features,structures, or characteristics described may be combined in various waysin one or more implementations.

Provided herein is a novel type of wide-spectrum MBLs inhibitor—Bi(III)compounds or the pharmaceutically acceptable salts thereof, thatmodulate MBLs activity via a metal replacement mechanism. Providedherein are methods to treat infection from MBL-producing bacteria byadministering the MBLs inhibitors with β-lactam antibiotics.

Provided herein are methods of preparing Bi(III) compounds byrepositioning of Bi(III) compounds and coordinating Bi(III) to N, O, orS containing ligands. Examples of the ligands of Bi(III) include, butare not limited to,

Provided herein are composition comprising a Bi(III) compound orpharmaceutically acceptable salt thereof. In certain embodiments, theBi(III) compound or pharmaceutically acceptable salt thereof is aBi(III) complex. In certain embodiments, the Bi(III) complex includesthe complexes listed in Table 1.

In certain embodiments, the β-lactam antibiotic and the MBLs inhibitorhave a molar ratio ranging from 1:16, 2:15, 3:14, 4:13; 5:12, 6:11,7:10, 8:9, 9:8, 10:7, 11:6, 12:5, 13:4, 14:3, 14:3, 15:2, 16:1 by weight(w/w). In certain embodiments, the β-lactam antibiotic and the MBLsinhibitor have a molar ratio ranging from 1:16 to 16:1 by weight (w/w).

β-lactam antibiotics are a broad class of antibiotics which arecharacterized by their four-membered, nitrogen-containing beta-lactamring at the core of their structure. Examples of β-lactam antibioticsinclude, but not limited to penicillin derivatives (penams),cephalosporins (cephems), carbapenems, and monobactams.

In one embodiment, the β-lactam antibiotic is meropenem (“MER”) whichbelongs to carbapenem and has extended-broad-spectrum antibacterialactivity against a wide variety of bacteria. Similar to other β-lactamantibiotics, the beta-lactam ring portion of MER binds to differentDD-transpeptidases, viz, penicillin-binding proteins (“PBPs”) in cellmembrane rendering them unable to perform their roles in synthesis ofthe peptidoglycan layer of bacterial cell wall. This results in thebacterial death owing to osmotic instability or autolysis. The structureof MER is represented by the following formula:

The mechanism of action of Bi(III) is unveiled via X-ray crystallographywhich showed that one bismuth ion replaced two zinc ions, leading toinactivation of the MBLs. Based on the fact that MBLs are inactivated inthese bacteria through the same mechanism, Bi(III) compounds or thepharmaceutically acceptable salts thereof disclosed herein serve aswide-spectrum inhibitors of MBLs. In some embodiments, Bi(III) compoundsor the pharmaceutically acceptable salt thereof, exhibited no ornegligible in vitro cytotoxicity to human cells. In some embodiments,the bismuth compounds showed good inhibitory activity on MBLs. Upon theuse of Bi(III) compounds or the pharmaceutically acceptable saltthereof, β-lactam antibiotic shows revived antimicrobial activity andprevents the growth or kills MBL-producing bacteria at clinicallyrelevant susceptible range. In one embodiment, the combination of MERand CBS exerts synergistic effect in the treatment of in vitro bacterialinfection. In certain embodiments, MER show boosted efficacy in an invivo murine peritonitis infection model when co-administered with CBS.In certain embodiments, the antibacterial efficacy of the Bi(III)complexes or the pharmaceutically acceptable salt thereof is enhanced by2-4, 4-6, 6-8 folds as compared to antibiotics that are without theBi(III) complexes or the pharmaceutically acceptable salt thereof. Incertain embodiments, the antibacterial efficacy of MER is enhanced by4-8 folds in the presence of Bi(III) compound or the pharmaceuticallyacceptable salt thereof as compared to MER alone.

In some embodiments, Bi(III) center of different complexes exist in theform of monomer, dimer or polymer in solution. The Bi(III) coordinationcore usually is negatively charged, thus requires at least onecounter-cation to achieve electric neutrality. In certain embodiments,pharmaceutically acceptable salts thereof include those generated fromcharged bismuth complexes and counter cation and/or anion.

In certain embodiments, MBLs require Zn(II) for the catalysis and areable to hydrolyze β-lactam antibiotics. Examples include, but notlimited to, BCII, CcrA, IMP, VIM, NDM and DIM. In some embodiments, theβ-lactamase includes NDM-1, VIM-2, and IMP-4.

In some embodiments, the inhibitors refer to Bi(III) compounds or thepharmaceutically acceptable salts thereof.

Bi(III) compounds or the pharmaceutically acceptable salts thereofdescribed herein relate to stable Bi(III) compounds or thepharmaceutically acceptable salts thereof. Bi(III) compounds comprisecomplexes with Bi(III) coordinated to but not limited to N, O or Scontaining ligands. The ligands involved include, but are not limitedto,

Given that cells exposed to a cytotoxic substance may undergo necrosis,where they lose membrane integrity and die rapidly due to cell lysis; orthey may discontinue growing and proliferating; or they may initiateapoptosis, which is a genetic program of controlled cell death. In oneembodiment, XTT assay is used to examine the cytotoxicity of Bi(III)complexes to human cells. The human cell lines include, but not limitedto human hepatocyte cell line (MIHA).

In some embodiments, examples of MBL producing bacteria include, but notlimited to some enterobacterial strains, such as E. coli (NDM-1⁺), E.coli (VIM-2⁺), E. coli (IMP-4⁺), K. pneumonia (NDM-1⁺) and C. freundii(NDM-1⁺).

In one specific embodiment, the clinically relevant susceptible range ofcarbapenem towards Enterobacteriaceae is ≤2 μg mL−1 according to thecriteria of European Committee on Antimicrobial Susceptibility Testing(EUCAST).

In some specific embodiments, the synergistic effect is quantified bythe calculation of fractional inhibitory concentration index (FICI).

In one specific embodiment, madin-darby canine kidney (MDCK) cells arechosen as the context for in vitro bacterial infection experiment. Inone specific embodiment, BALB/c mice are chosen as the context for invivo murine infection experiment.

In some embodiments, the mechanism of the inhibition of purified NDM-1was investigated by determining the ‘metal content’ and the ‘enzymeactivity’ of NDM-1 protein. About a quarter to 30% of proteins in humansand microbes are found to bind to various important metal ions to exerttheir in vivo functions and they are generally regarded as‘metallo-proteins’. In some embodiments, both the Bi(III) and Zn(II)contents were studied.

In one embodiment, the purified NDM-1 was crystallized and incubatedwith Bi(III) compounds followed by X-ray diffraction experiments togenerate a three dimensional structural data of the protein. The bindingof Bi(III) to the protein was then observed using suitable structuralbiology computer software as described in the example section below.

5.2 Combination Therapy

The compounds as described herein may be optionally delivered with otherantibacterial agents in the form of antibacterial cocktails, orindividually, yet close enough in time to have a synergistic effect onthe treatment of the infection. An antibacterial cocktail is a mixtureof any one of the compounds described herein with another antibacterialdrug. In one embodiment, a common administration vehicle (e.g., tablet,implants, injectable solution, injectable liposome solution, etc.) isused in for the compound as described herein and other antibacterialagent(s).

5.3 Pharmaceutical Compositions

The compound disclosed herein can be formulated into pharmaceuticalcompositions using methods available in the art and those disclosedherein. Such compounds can be used in some embodiments to enhancedelivery of the compound to the subject.

The methods provided herein encompass administering pharmaceuticalcompositions containing at least one compound as described herein, ifappropriate in the salt form, either used alone or in the form of acombination with one or more compatible and pharmaceutically acceptablecarriers, such as diluents or adjuvants, or with another antibacterialagent. In certain embodiments, the second agent can be formulated orpackaged with the compound provided herein, according to those of skillin the art, such co-formulation should not interfere with the activityof either agent or the method of administration. In certain embodiments,the compound provided herein and the second agent are formulatedseparately. They can be packaged together, or packaged separately, forthe convenience of the practitioner of skill in the art. In clinicalpractice the active agents provided herein may be administered by anyconventional route, in particular orally, parenterally, rectally or byinhalation (e.g. in the form of aerosols). In certain embodiments, thecompound provided herein is administered orally. Use may be made, assolid compositions for oral administration, of tablets, pills, hardgelatin capsules, powders or granules. In these compositions, the activeproduct is mixed with one or more inert diluents or adjuvants, such assucrose, lactose or starch. These compositions can comprise substancesother than diluents, for example a lubricant, such as magnesiumstearate, or a coating intended for controlled release. Use may be made,as liquid compositions for oral administration, of solutions which arepharmaceutically acceptable, suspensions, emulsions, syrups and elixirscontaining inert diluents, such as water or liquid paraffin. Thesecompositions can also comprise substances other than diluents, forexample wetting, sweetening or flavoring products. The compositions forparenteral administration can be emulsions or sterile solutions. Use maybe made, as solvent or vehicle, of propylene glycol, a polyethyleneglycol, vegetable oils, in particular olive oil, or injectable organicesters, for example ethyl oleate. These compositions can also containadjuvants, in particular wetting, isotonizing, emulsifying, dispersingand stabilizing agents. Sterilization can be carried out in severalways, for example using a bacteriological filter, by radiation or byheating. They can also be prepared in the form of sterile solidcompositions which can be dissolved at the time of use in sterile wateror any other injectable sterile medium. The compositions for rectaladministration are suppositories or rectal capsules which contain, inaddition to the active principle, excipients such as cocoa butter,semi-synthetic glycerides or polyethylene glycols. The compositions canalso be aerosols. For use in the form of liquid aerosols, thecompositions can be stable sterile solutions or solid compositionsdissolved at the time of use in sterile water, in saline or any otherpharmaceutically acceptable vehicle. For use in the form of dry aerosolsintended to be directly inhaled, the active principle is finely dividedand combined with a water-soluble solid diluent or vehicle, for exampledextran, mannitol or lactose. In one embodiment, a composition providedherein is a pharmaceutical composition or a single unit dosage form.Pharmaceutical compositions and single unit dosage forms provided hereincomprise a therapeutically effective amount of one or more therapeuticagents (e.g., a compound provided herein, or other prophylactic ortherapeutic agent), and one or more pharmaceutically acceptable carriersor excipients. The term “carrier” includes a diluent, adjuvant (e.g.,Freund's adjuvant (complete and incomplete)), excipient, or vehicle withwhich the therapeutic is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water can be used asa carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well-known to those skilled inthe art of pharmacy, and non limiting examples of suitable excipientsinclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a subjectand the specific active ingredients in the dosage form. The compositionor single unit dosage form, if desired, can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents. Lactose freecompositions provided herein can comprise excipients that are well knownin the art and are listed, for example, in the U.S. Pharmocopia. Ingeneral, lactose free compositions comprise an active ingredient, abinder/filler, and a lubricant in pharmaceutically compatible andpharmaceutically acceptable amounts. Exemplary lactose free dosage formscomprise an active ingredient, microcrystalline cellulose, pregelatinized starch, and magnesium stearate. Further encompassed hereinare anhydrous pharmaceutical compositions and dosage forms comprisingactive ingredients, since water can facilitate the degradation of somecompounds. Anhydrous pharmaceutical compositions and dosage formsprovided herein can be prepared using anhydrous or low moisturecontaining ingredients and low moisture or low humidity conditions. Ananhydrous pharmaceutical composition should be prepared and stored suchthat its anhydrous nature is maintained. Accordingly, anhydrouscompositions can be packaged using materials known to prevent exposureto water such that they can be included in suitable formulary kits.Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs. The pharmaceutical compositions andsingle unit dosage forms can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Such compositions and dosage forms will contain a prophylacticallyor therapeutically effective amount of a prophylactic or therapeuticagent, in certain embodiments, in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the subject. The formulation should suit the mode ofadministration. In a certain embodiment, the pharmaceutical compositionsor single unit dosage forms are sterile and in suitable form foradministration to a subject, for example, an animal subject, such as amammalian subject, for example, a human subject.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude, but are not limited to, parenteral, e.g., intravenous,intradermal, subcutaneous, intramuscular, subcutaneous, oral, buccal,sublingual, inhalation, intranasal, transdermal, topical, transmucosal,intra-tumoral, intra-synovial and rectal administration. In a specificembodiment, the composition is formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenous,subcutaneous, intramuscular, oral, intranasal or topical administrationto human beings. In an embodiment, a pharmaceutical composition isformulated in accordance with routine procedures for subcutaneousadministration to human beings. Typically, compositions for intravenousadministration are solutions in sterile isotonic aqueous buffer. Wherenecessary, the composition may also include a solubilizing agent and alocal anesthetic such as lignocamne to ease pain at the site of theinjection. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; ointments;cataplasms (poultices); pastes; powders; dressings; creams; plasters;solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to asubject, including suspensions (e.g., aqueous or non aqueous liquidsuspensions, oil in water emulsions, or a water in oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a subject; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a subject.The composition, shape, and type of dosage forms provided herein willtypically vary depending on their use. For example, a dosage form usedin the initial treatment of bacterial infection may contain largeramounts of one or more of the active ingredients it comprises than adosage form used in the maintenance treatment of the same infection.Similarly, a parenteral dosage form may contain smaller amounts of oneor more of the active ingredients it comprises than an oral dosage formused to treat the same disease or disorder. These and other ways inwhich specific dosage forms encompassed herein will vary from oneanother will be readily apparent to those skilled in the art. See, e.g.,Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing, EastonPa. (2000). Typical dosage forms comprise a compound provided herein, ora pharmaceutically acceptable salt, solvate or hydrate thereof liewithin the range of from about 0.1 mg to about 1000 mg per day, given asa single once-a-day dose in the morning or as divided doses throughoutthe day taken with food. Particular dosage forms can have about 0.1,0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0,100, 200, 250, 500 or 1000 mg of the active compound. Oral Dosage FormsPharmaceutical compositions that are suitable for oral administrationcan be presented as discrete dosage forms, such as, but are not limitedto, tablets (e.g., chewable tablets), caplets, capsules, and liquids(e.g., flavored syrups). Such dosage forms contain predetermined amountsof active ingredients, and may be prepared by methods of pharmacy wellknown to those skilled in the art. See generally, Remington'sPharmaceutical Sciences, 20th ed., Mack Publishing, Easton Pa. (2000).

In certain embodiments, provided herein is a hand sanitizing compositioncomprising the compounds disclosed herein. In certain embodiments,provided herein is a lotion comprising the compounds as disclosedherein.

5.4 Dosage and Unit Dosage Forms

In human therapeutics, the doctor will determine the posology which heconsiders most appropriate according to a preventive or curativetreatment and according to the age, weight, stage of the infection andother factors specific to the subject to be treated. In certainembodiments, doses are from about 1 to about 1000 mg per day for anadult, or from about 5 to about 250 mg per day or from about 10 to 50 mgper day for an adult. In certain embodiments, doses are from about 5 toabout 400 mg per day or 25 to 200 mg per day per adult. In certainembodiments, dose rates of from about 50 to about 500 mg per day arealso contemplated.

In further aspects, provided are methods of treating or preventing abacterial infection in a subject by administering, to a subject in needthereof, an effective amount of a compound provided herein, or apharmaceutically acceptable salt thereof. The amount of the compound orcomposition which will be effective in the prevention or treatment of adisorder or one or more symptoms thereof will vary with the nature andseverity of the infection, and the route by which the active ingredientis administered. The frequency and dosage will also vary according tofactors specific for each subject depending on the specific therapy(e.g., therapeutic or prophylactic agents) administered, the severity ofthe infection, the route of administration, as well as age, body,weight, response, and the past medical history of the subject. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems.

In certain embodiments, exemplary doses of a composition includemilligram or microgram amounts of the active compound per kilogram ofsubject or sample weight (e.g., about 10 micrograms per kilogram toabout 50 milligrams per kilogram, about 100 micrograms per kilogram toabout 25 milligrams per kilogram, or about 100 microgram per kilogram toabout 10 milligrams per kilogram). For compositions provided herein, incertain embodiments, the dosage administered to a subject is 0.140 mg/kgto 3 mg/kg of the subject's body weight, based on weight of the activecompound. In certain embodiments, the dosage administered to a subjectis between 0.20 mg/kg and 2.00 mg/kg, or between 0.30 mg/kg and 1.50mg/kg of the subject's body weight.

In certain embodiments, the recommended daily dose range of acomposition provided herein for the conditions described herein liewithin the range of from about 0.1 mg to about 1000 mg per day, given asa single once-a-day dose or as divided doses throughout a day. In oneembodiment, the daily dose is administered twice daily in equallydivided doses. In certain embodiments, a daily dose range should be fromabout 10 mg to about 200 mg per day, in other embodiments, between about10 mg and about 150 mg per day, in further embodiments, between about 25and about 100 mg per day. It may be necessary to use dosages of theactive ingredient outside the ranges disclosed herein in some cases, aswill be apparent to those of ordinary skill in the art. Furthermore, itis noted that the clinician or treating physician will know how and whento interrupt, adjust, or terminate therapy in conjunction with subjectresponse.

Different therapeutically effective amounts may be applicable fordifferent infections, as will be readily known by those of ordinaryskill in the art. Similarly, amounts sufficient to prevent, manage,treat or ameliorate such infections, but insufficient to cause, orsufficient to reduce, adverse effects associated with the compositionprovided herein are also encompassed by the above described dosageamounts and dose frequency schedules. Further, when a subject isadministered multiple dosages of a composition provided herein, not allof the dosages need be the same. For example, the dosage administered tothe subject may be increased to improve the prophylactic or therapeuticeffect of the composition or it may be decreased to reduce one or moreside effects that a particular subject is experiencing.

In certain embodiment, the dosage of the composition provided herein,based on weight of the active compound, administered to prevent, treat,manage, or ameliorate an infection, or one or more symptoms thereof in asubject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject's body weight. Inanother embodiment, the dosage of the composition or a compositionprovided herein administered to prevent, treat, manage, or ameliorate aninfection, or one or more symptoms thereof in a subject is a unit doseof 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg,0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg,1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5mg, 1 mg to 5 mg, or 1 mg to 2.5 mg. In certain embodiments, treatmentor prevention can be initiated with one or more loading doses of acompound or composition provided herein followed by one or moremaintenance doses. In such embodiments, the loading dose can be, forinstance, about 60 to about 400 mg per day, or about 100 to about 200 mgper day for one day to five weeks. The loading dose can be followed byone or more maintenance doses. In certain embodiments, each maintenancedoes is, independently, about from about 10 mg to about 200 mg per day,between about 25 mg and about 150 mg per day, or between about 25 andabout 80 mg per day. Maintenance doses can be administered daily and canbe administered as single doses, or as divided doses. In certainembodiments, a dose of a compound or composition provided herein can beadministered to achieve a steady-state concentration of the activeingredient in blood or serum of the subject. The steady-stateconcentration can be determined by measurement according to techniquesavailable to those of skill or can be based on the physicalcharacteristics of the subject such as height, weight and age. Incertain embodiments, a sufficient amount of a compound or compositionprovided herein is administered to achieve a steady-state concentrationin blood or serum of the subject of from about 300 to about 4000 ng/mL,from about 400 to about 1600 ng/mL, or from about 600 to about 1200ng/mL. In some embodiments, loading doses can be administered to achievesteady-state blood or serum concentrations of about 1200 to about 8000ng/mL, or about 2000 to about 4000 ng/mL for one to five days. Incertain embodiments, maintenance doses can be administered to achieve asteady-state concentration in blood or serum of the subject of fromabout 300 to about 4000 ng/mL, from about 400 to about 1600 ng/mL, orfrom about 600 to about 1200 ng/mL. In certain embodiments,administration of the same composition may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or 6 months. In other embodiments, administration of the sameprophylactic or therapeutic agent may be repeated and the administrationmay be separated by at least at least 1 day, 2 days, 3 days, 5 days, 10days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6months. In certain aspects, provided herein are unit dosages comprisinga compound, or a pharmaceutically acceptable salt thereof, in a formsuitable for administration. Such forms are described in detail above.In certain embodiments, the unit dosage comprises 1 to 1000 mg, 5 to 250mg or 10 to 50 mg active ingredient. In particular embodiments, the unitdosages comprise about 1, 5, 10, 25, 50, 100, 125, 250, 500 or 1000 mgactive ingredient. Such unit dosages can be prepared according totechniques familiar to those of skill in the art. The dosages of thesecond agents are to be used in the combination therapies providedherein. In certain embodiments, dosages lower than those which have beenor are currently being used to prevent or treat bacterial infection areused in the combination therapies provided herein. The recommendeddosages of second agents can be obtained from the knowledge of those ofskill. For those second agents that are approved for clinical use,recommended dosages are described in, for example, Hardman et al., eds.,1996, Goodman & Gilman's The Pharmacological Basis Of Basis OfTherapeutics 9.sup.th Ed, Mc-Graw-Hill, New York; Physician's DeskReference (PDR) 57.sup.th Ed., 2003, Medical Economics Co., Inc.,Montvale, N.J., which are incorporated herein by reference in itsentirety.

In various embodiments, the therapies (e.g., a compound provided hereinand a second agent in a combination therapy) are administered less than5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1hour apart, at about 1 to about 2 hours apart, at about 2 hours to about3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In variousembodiments, the therapies are administered no more than 24 hours apartor no more than 48 hours apart. In certain embodiments, two or moretherapies are administered within the same patient visit. In otherembodiments, the compound provided herein and the second agent areadministered concurrently. In other embodiments, the compound providedherein and the second agent are administered at about 2 to 4 days apart,at about 4 to 6 days apart, at about 1 week part, at about 1 to 2 weeksapart, or more than 2 weeks apart. In certain embodiments,administration of the same agent may be repeated and the administrationsmay be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. Inother embodiments, administration of the same agent may be repeated andthe administration may be separated by at least at least 1 day, 2 days,3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3months, or 6 months. In certain embodiments, a compound provided hereinand a second agent are administered to a patient, for example, a mammal,such as a human, in a sequence and within a time interval such that thecompound provided herein can act together with the other agent toprovide an increased benefit than if they were administered otherwise.For example, the second active agent can be administered at the sametime or sequentially in any order at different points in time; however,if not administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeutic orprophylactic effect. In one embodiment, the compound provided herein andthe second active agent exert their effect at times which overlap. Eachsecond active agent can be administered separately, in any appropriateform and by any suitable route. In other embodiments, the compoundprovided herein is administered before, concurrently or afteradministration of the second active agent. In certain embodiments, thecompound provided herein and the second agent are cyclicallyadministered to a patient. Cycling therapy involves the administrationof a first agent (e.g., a first prophylactic or therapeutic agents) fora period of time, followed by the administration of a second agentand/or third agent (e.g., a second and/or third prophylactic ortherapeutic agents) for a period of time and repeating this sequentialadministration. Cycling therapy can reduce the development of resistanceto one or more of the therapies, avoid or reduce the side effects of oneof the therapies, and/or improve the efficacy of the treatment. Incertain embodiments, the compound provided herein and the second activeagent are administered in a cycle of less than about 3 weeks, about onceevery two weeks, about once every 10 days or about once every week. Onecycle can comprise the administration of a compound provided herein andthe second agent by infusion over about 90 minutes every cycle, about 1hour every cycle, about 45 minutes every cycle. Each cycle can compriseat least 1 week of rest, at least 2 weeks of rest, at least 3 weeks ofrest. The number of cycles administered is from about 1 to about 12cycles, more typically from about 2 to about 10 cycles, and moretypically from about 2 to about 8 cycles. In other embodiments, coursesof treatment are administered concurrently to a patient, i.e.,individual doses of the second agent are administered separately yetwithin a time interval such that the compound provided herein can worktogether with the second active agent. For example, one component can beadministered once per week in combination with the other components thatcan be administered once every two weeks or once every three weeks. Inother words, the dosing regimens are carried out concurrently even ifthe therapeutics are not administered simultaneously or during the sameday. The second agent can act additively or synergistically with thecompound provided herein. In one embodiment, the compound providedherein is administered concurrently with one or more second agents inthe same pharmaceutical composition. In another embodiment, a compoundprovided herein is administered concurrently with one or more secondagents in separate pharmaceutical compositions. In still anotherembodiment, a compound provided herein is administered prior to orsubsequent to administration of a second agent. Also contemplated areadministration of a compound provided herein and a second agent by thesame or different routes of administration, e.g., oral and parenteral.In certain embodiments, when the compound provided herein isadministered concurrently with a second agent that potentially producesadverse side effects including, but not limited to, toxicity, the secondactive agent can advantageously be administered at a dose that fallsbelow the threshold that the adverse side effect is elicited.

5.5 Patient Population

In some embodiments, a subject treated for infection in accordance withthe methods provided herein is a human who has or is diagnosed with aninfection. In other embodiments, a subject treated for infection inaccordance with the methods provided herein is a human predisposed orsusceptible to infection. In some embodiments, a subject treated forinfection in accordance with the methods provided herein is a human atrisk of developing infection.

In one embodiment, a subject treated for infection in accordance withthe methods provided herein is a human infant. In another embodiment, asubject treated for infection in accordance with the methods providedherein is a human toddler. In another embodiment, a subject treated forinfection in accordance with the methods provided herein is a humanchild. In another embodiment, a subject treated for infection inaccordance with the methods provided herein is a human adult. In anotherembodiment, a subject treated for infection in accordance with themethods provided herein is a middle-aged human. In another embodiment, asubject treated for infection in accordance with the methods providedherein is an elderly human.

In some embodiments, a subject treated for infection in accordance withthe methods provided herein that has a recurrence of the infection.

In certain embodiments, a subject treated for infection in accordancewith the methods provided herein is a human that is about 1 to about 5years old, about 5 to 10 years old, about 10 to about 18 years old,about 18 to about 30 years old, about 25 to about 35 years old, about 35to about 45 years old, about 40 to about 55 years old, about 50 to about65 years old, about 60 to about 75 years old, about 70 to about 85 yearsold, about 80 to about 90 years old, about 90 to about 95 years old orabout 95 to about 100 years old, or any age in between. In a specificembodiment, a subject treated for infection in accordance with themethods provided herein is a human that is 18 years old or older. In aparticular embodiment, a subject treated for infection in accordancewith the methods provided herein is a human child that is between theage of 1 year old to 18 years old. In a certain embodiment, a subjecttreated for infection in accordance with the methods provided herein isa human that is between the age of 12 years old and 18 years old. In acertain embodiment, the subject is a male human. In another embodiment,the subject is a female human. In one embodiment, the subject is afemale human that is not pregnant or is not breastfeeding. In oneembodiment, the subject is a female that is pregnant or will/mightbecome pregnant, or is breast feeding.

In some embodiments, a subject treated for infection in accordance withthe methods provided herein is administered a pharmaceutical compositionthereof, or a combination therapy before any adverse effects orintolerance to therapies.

In some embodiments, a subject treated for infection accordance with themethods provided herein is a human that has established resistance toprevious antibiotic therapies other than treatment with the presentlydisclosed therapy. In certain embodiments, a subject treated forinfection in accordance with the methods provided herein is a humanalready receiving one or more conventional antibacterial therapies.Among these patients are patients who have developed infections that areresistant to antibiotics and patients with recurring infections despitetreatment with existing therapies. In certain embodiments, the patientshave been previously treated with β-lactum antibiotics. In certainembodiments, the patients have been previously treated with carbapenem.In certain embodiments, the patients have been previously treated withmeropenem, imipenem, ertapenem, doripenem, biapenem, faropenem,panipenem, razupenem, tebipenem, tomopenem or a combination thereof. Incertain embodiments, the patients have developed infections that areresistant to β-lactum antibiotics. In certain embodiments, the patientshave developed infections that are resistant to carbapenem. In certainembodiments, the patients have developed infections that are resistantto meropenem, imipenem, ertapenem, doripenem, biapenem, faropenem,panipenem, razupenem, tebipenem, tomopenem or a combination thereof.

5.6 Kits

Also provided are kits for use in methods of treatment of a bacterialinfection. The kits can include a compound or composition providedherein, a second agent or composition, and instructions providinginformation to a health care provider regarding usage for treating theinfection. Instructions may be provided in printed form or in the formof an electronic medium such as a floppy disc, CD, or DVD, or in theform of a website address where such instructions may be obtained. Aunit dose of a compound or composition provided herein, or a secondagent or composition, can include a dosage such that when administeredto a subject, a therapeutically or prophylactically effective plasmalevel of the compound or composition can be maintained in the subjectfor at least 1 days. In some embodiments, a compound or composition canbe included as a sterile aqueous pharmaceutical composition or drypowder (e.g., lyophilized) composition. In some embodiments, suitablepackaging is provided. As used herein, “packaging” includes a solidmatrix or material customarily used in a system and capable of holdingwithin fixed limits a compound provided herein and/or a second agentsuitable for administration to a subject. Such materials include glassand plastic (e.g., polyethylene, polypropylene, and polycarbonate)bottles, vials, paper, plastic, and plastic-foil laminated envelopes andthe like. If e-beam sterilization techniques are employed, the packagingshould have sufficiently low density to permit sterilization of thecontents.

The kits described herein contain one or more containers, which containcompounds, signaling entities, biomolecules and/or particles asdescribed. The kits also contain instructions for mixing, diluting,and/or administrating the compounds. The kits also include othercontainers with one or more solvents, surfactants, preservative and/ordiluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well ascontainers for mixing, diluting or administering the components to thesample or to the patient in need of such treatment.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the powder may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are sued, the liquidform may be concentrated or ready to use. The solvent will depend on thecompound and the mode of use or administration. Suitable solvents fordrug compositions are well known and are available in the literature.The solvent will depend on the compound and the mode of use oradministration.

The kits comprise a carrier being compartmentalized to receive in closeconfinement one or more container such as vials, tubes, and the like,each of the container comprising one of the separate elements to be usedin the method. For example, one of the container may comprise a positivecontrol in an assay. Additionally, the kit may include containers forother components, for example, buffers useful in the assay.

5.7 Method of Treatment

Provided herein is a method of reducing growth of a bacteria byadministering one or more compounds provided herein. The methodcomprises contacting a cell with one or more compounds as describedherein in an amount effective to reduce the growth of bacteria. Providedherein are methods for treating a bacterial infection in a subject byadministering to a subject in need thereof the compounds describedherein in an amount effective to reduce the growth of bacteria.

5.8 Methods of Coating a Medical Device

The compounds that are provided herein can be used to treat a variety ofmedical devices, such as catheters, as well as industrial surfaces.Bacteria can form biofilm on intravascular catheters and other medicalimplants. These biofilms enhance antimicrobial resistance and can renderinfections refractory to antibacterial therapy. Persistence of aninfection can necessitate removal of the device, which can beundesirable or even life threatening. Therefore, provided herein is amethod of coating a medical device using the compounds described hereinon the materials or surfaces of the medical device that mitigate orprevent bacterial colonization or infection with subsequent biofilmformation. The method comprises applying the composition describedherein on the surface of a medical device. In certain embodiments, thecomposition adheres to the surface of a medical device. In certainembodiments, the composition is coated in the medical device.

In certain embodiments, provided herein is a composition comprising apaint and one or more compounds as described herein. Also provided is amethod of modifying a surface of a medical device. The method comprisesproviding one or more coatings of a composition, comprising one or morecompounds as described herein, to at least a portion of the surface of amedical device to form a coated surface region. In certain embodiments,the medical device is an implantable medical device. In certainembodiments, the industrial surface is stainless steel. In certainembodiments, the industrial surface is plastic. In certain embodiments,the industrial surface is a surgical table. In certain embodiments, themedical device is a surgical instrument.

The following Examples illustrate the synthesis and use ofrepresentative compounds provided herein. These examples are notintended, nor are they to be construed, as limiting the scope of theclaimed subject matter. It will be clear that the scope of subjectmatter may be practiced otherwise than as particularly described herein.Numerous modifications and variations of the subject matter are possiblein view of the teachings herein and, therefore, are within the scope theclaimed subject matter.

6. EXAMPLES 6.1. Example 1: Preparation of the Exemplary BismuthComplexes 6.1.1 Synthesis of Complex 1, 2, 3 and 4

Complexes 1, 2, 3 and 4 were synthesized. Take the synthesis of complex1 for instance, 5,10,15,20-tetraphenyl-21H-23H-porphine (L1, 0.20 mmol)was added to pyridine (50 mL) and the temperature was increased untilthe purple solution was refluxing. Bi(NO₃)₃.5H₂O (2.10 mmol) was thenadded and the solution was left to reflux for 3 hours. MoreBi(NO₃)₃.5H₂O (4.10 mmol) was added and the solution was left to refluxfor a further 5 hours. The resulting dark-green solution was then leftto cool to room temperature before the bulk of the pyridine solvent wasremoved via rotary evaporation. The resulting green mixture was thenleft to dry overnight under vacuum to remove any remaining solvents. Thegreen solid obtained was then washed with chloroform (20 mL) and rotaryevaporated to ensure that all pyridine was removed. This was repeatedfor 5 times followed by overnight drying under vacuum. Dark green solidwas then purified on a silica gel column. The compound was loaded ontothe column using a dry method and the product was obtained by elutingthe column with chloroform and methanol in a ratio of 20:1.

6.1.2 Synthesis of Complex 5

Bi(NO₃)₃.5H₂O (1.00 mmol) was added to deionized water (50 mL) resultingin a cloudy white solution. The temperature was increased until thesolution was refluxing and had turned colorless. At this point,2-picolinic acid (3.00 mmol) (L5) was added and the resulting colorlesssolution was left to reflux for 2 hours. The solution was then allowedto cool to room temperature before being filtered under vacuum to removeany insoluble impurities. The colorless filtrate was then decanted intoa beaker, covered and allowed to stand at room temperature for 2 daysafter that small amounts of small, colorless crystals of complex,suitable for x-ray analysis, were obtained. The crystalline product wasthen collected via vacuum filtration.

6.1.3 Synthesis of Complex 7

Bismuth complexation with thiosalicylic acid (L7) was prepared bydissolving Bi(NO₃)₃.5H₂O (1.00 mmol) and thiosalicylic acid (2.50 mmol)in DMSO (15 mL), followed by sonication till dissolving. The mixture wasstirred at room temperature for 0.5 hour and then extracted by water andethyl acetate. The final product was obtained by recrystallizing andwashed by water and saturated NaCl solution and then dried in vacuum.

6.1.4 Synthesis of Complex 8

Bi(NO₃)₃.5H₂O (1.00 mmol) was refluxed in deionised water (15 mL) untilsolution turned colorless. 2-hydroxynicotinic acid (1.00 mmol) (L8) wasadded and the mixture was refluxed for an overnight. Any remaining solidwas filtered off and the filtrate was left at 4° C. for several days. Asmall yield of off-white solid formed and was washed with small volumeof deionized water. This left a white insoluble solid and a yellowaqueous solution. The solution was left for crystal formation.

6.1.5 Synthesis of Complex 9

Bi(NO₃)₃.5H₂O (1.00 mmol) was dissolved in ethanol (60 mL), withstirring, at room temperature for 0.5 hour, followed by the addition of8-hydroxyquinoline (3.00 mmol) (L9). The solution, which immediatelyturned yellow upon addition of the ligand, was left to reflux for 3hours after that it was left to cool to room temperature. The cloudyyellow solution was then filtered under vacuum and the clear, yellowfiltrate was collected. Triethylamine and deionized water were thenadded to cause immediate precipitation of a yellow solid. The solutionwas again vacuum filtered and the yellow powder was left to dry undervacuum overnight.

6.1.6 Synthesis of Complex 11 and 12

Complexes 11 and 12 were synthesized via a generic method. Take thesynthesis of complex 12 for instance, firstly (BiO)₂CO₃ (2.50 mmol) wasslowly added to hot EDTA-water solution (100 mL). The mixture wasrefluxed for 4 hours on oil bath and then filtered while hot. Thefiltrate was settled at 4° C. for one or two days and afforded smallcrystal by slow evaporation.

6.1.7 Synthesis of Complex 13

Bismuth complex with tetrabromocatechol (L13) was prepared by dissolvingBi(NO₃)₃.5H₂O (1.00 mmol) and L13 (1.00 mmol) in DMSO (20 mL) andstirring at room temperature for 1 hour. The solution was then extractedby water and ethyl acetate, followed by evaporation. The final productwas washed by water and saturated NaCl solution and then dried invacuum.

6.1.8 Synthesis of Complex 15

BiCl₃ (1.00 mmol) dissolved in methanol (15 mL), followed by addition ofHCl (37%, 50 μL). Captopril (3.00 mmol) (L15) was added and mixture wasstirred overnight at room temperature at pH 2. Sodium hydroxide wasadded to increase pH to 10. The solution changed its colour to orange.Methanol was removed in vacuum, and the resulting solid was filtered andwashed with minimum volume ethanol.

6.1.9 Synthesis of Complex 16

Bi(NO₃)₃.5H₂O (1.00 mmol) and 2,2′-bipyridine (2.00 mmol) (L16) weredissolved, with gentle heating, in a minimum amount of DMSO. Once fullydissolved, the warm colorless solution was filtered and the colorlessfiltrate was left to stand at room temperature for 14 days to yield asmall amount of pale pink crystals, suitable for x-ray analysis. Thecrystalline product was then collected via vacuum filtration.

6.1.10 Synthesis of Complex 17

Bi(NO₃)₃.5H₂O (1.00 mmol) and reduced form of glutathione (2.00 mmol)(L17) were dissolved in deionized water (20 mL), sonicated and left tostir at room temperature for 0.5 hour. The solution was then filtered toremove any insoluble impurities and then slowly rotary evaporated toremove most water. Then the wet sample was left in freeze drierovernight and collect solid product then.

6.1.11 Synthesis of Complex 20

BiCl₃ (1.00 mmol) dissolved in methanol (15 mL) containing penicillamine(3.00 mmol) (L20). Color change from colorless to yellow occurred.Mixture was stirred overnight at room temperature after which any solidwas removed by vacuum filtration. Solvent was removed from the resultingfiltrate and a yellow oil formed. This was left at 4° C. for severaldays resulting in a yellow solid. The solid was recrystallized frommethanol.

6.1.12 Synthesis of Complex 21

Bi(NO₃)₃.5H₂O (0.50 mmol) and N-acetyl-L-cysteine (1.50 mmol) (L21) weredissolved in deionized water (15 mL) and left to stir at roomtemperature for 1 hour. NaOH (3 mL, 1 M) was added to the resultingyellow solution to raise the pH to around 6. The solution was thenfiltered to remove any insoluble impurities and then rotary evaporatedto remove the water. The solid product was then washed with ethanol andvacuum filtered to remove extra NaNO₃.

Complex 6 (bismuth subsalicylate) and complex 14 (bismuth subgallate)were purchased from Alfa Aesar. Complex 10 (colloidal bismuthsubcitrate, CBS) and ranitidine bismuth citrate (RBC) were kindlyprovided by Livzon Pharmaceutical Group. The characteristic informationof these exemplary is shown in Table 1.

TABLE 1 Molecular Primary ESI-MS Assignment Complex formulaobserved(calculated) (m/z) (+ve/−ve) Solubility 1 C₄₄H₂₈BiN₄ 820.9(821.1) [Bi(L1)]⁺ DMSO 2 C₄₄H₂₈BiN₄O₄ 885.5 (885.2) [Bi(L2)]⁺ DMSO 3C₄₈H₃₆BiN₄ 877.8 (879.3) [Bi(L3)]⁺ DMSO 4 C₄₈H₃₆BiN₄O₄ 941.8 (943.2)[Bi(L4)]⁺ DMSO 5 C₁₈H₁₂BiN₂O₂ 496.9 (497.2) [Bi(L5)₂]⁺ 2% HCl 6 C₇H₅BiO₄— — Insoluble 7 C₁₄H₈BiO₄S₂ 513.3 (513.3) [Bi(L7)₂ + H]⁺ DMSO 8C₅H₁₁BiNO₄S•NaNO₃ 473.6 (474.2) [Bi(L8)(OH)₂ + NaNO₃]⁺ DMSO 9C₁₂H₁₀BiN₂O₄ 459.2 (459.9) [Bi(L9)₂ + 2H]⁺ DMSO 10 C₁₂H₈KBi₂O₁₄ 833.3(833.3) [Bi2(L10)₂ + K]⁻ H2O 11 C₆H₆BiNO₆•2H₂O 432.6 (432.1)[Bi(L11)(H₂O)₂ − 4H]⁻ H₂O 12 Bi-EDTA — — H₂O 13 C₁₂BiO₄Br₈ 1057.1(1056.3) [Bi(L13)₂]⁻ DMSO 14 C₇H₅BiO₆ — — Insoluble 15C₁₈H₂₆Bi₂N₂O₆S₂•KCl 922.4 (923.1) [Bi(L15)₂ + KCl]⁺ DMSO 16 C₂₀H₁₆BiN₆O₆645.1 (645.3) [Bi(L16)₂(NO₃)₂]⁺ DMSO 17 C₁₀H₁₅BiN₃O₆S 512.1 (512.1)[Bi(L17) − 2H]⁻ H₂O 18 C₆H₁₂BiN₃O₆ 430.9 (431.2) [Bi(L18)₃]⁺ H₂O 19C₉H₁₈BiN₃O₉Cl₂ 593.0 (592.1) [Bi(L19)₃]⁺ DMSO 20 C₅H₁₀BiNO₄S•NaNO₃ 473.6(474.2) [Bi(L20)(OH)₂ + NaNO₃]⁺ DMSO 21 C₁₅H₂₇BiN₃O₉S₃•NaNO₃ 784.1(783.5) [Bi(L21)₃ + (NaNO₃)₃]⁺ H₂O RBC — — — H₂O

TABLE 2 MBL Compounds IC₅₀ (μM) NDM-1 10 (CBS) 5.83 ± 0.53 12 3.86 ±0.95 21 17.69 ± 0.98  Bi(NO₃)₃ 1.89 ± 0.23 VIM-2 10 (CBS) 44.45 ± 3.94 15 6.17 ± 0.35 Bi(NO₃)₃ 2.45 ± 0.63

TABLE 3 Compound K_(m) V_(max) — 53.7 ± 9.5 0.85 ± 0.04 10 (CBS) 22.3 ±5.4 0.26 ± 0.01

6.2 Example 2: Cytotoxicity of Exemplary Bi(III) Complexes 6.2.1 XTTAssay

The growth and viability characteristics from the effect of bismuth onMIHA cells were measured using the cell proliferation kit II XTT assay(Roche Diagnostics, USA), according to the manufacturer's instruction.1-2×10⁴ cells/well were grown in flat-bottom 96-well plates (Costar,Corning Incorporated, NY, USA) in a final volume of 100 μl culturemedium per well for an overnight. Cells were then exposed to bismuthcompounds (1 to 100 μM) for 2 days. Those cells grown under culturemedium alone were used as a negative control. After a fixed incubationduration, 50 μl of XTT labeling mixture was added to each well, and thecells were incubated for 2 hours at 37° C. under a humidified atmosphereof 5% CO₂. The formation of formazan dyes, produced only by metabolicactive cells, was detected spectrophotometrically at 490 nm. From theXTT test result shown in FIG. 2, most exemplary bismuth complexes didnot exhibit or showed very low cytotoxicity against MIHA cells even at100 μM (the highest dose test) except for 9 and 13.

6.3 Example 3: In Vitro Mechanistic Studies of Enzyme Inhibition 6.3.1Construction of Wild-Type NDM-1 and VIM-2 Expression Vector

Full length NDM-1 protein comprises an N-terminal signal peptide(Met1-Pro28) for directing the protein to the periplasm of bacteriaafter protein translation and was subsequently cleaved during proteinmaturation process. The wild type expression vectors pET-28a-NDM-1 andpET-28a-VIM-2 were generated as described below. Polymerase chainreaction (PCR) was performed using KOD Hot Start DNA Polymerase(Novagen). The synthesized primer pair“NDM-1_29-270_WT_for/NDM-1_29-270_WT_rev” or“VIM-2_21-226_WT_for/VIM-2_21-226_WT_rev” (Life Technologies) wasrespectively used for amplifying the gene encoding NDM-1 protein(Gly29-Arg270) or VIM-2 protein (Ser21-Glu266). As a result, NdeI andEcoRI restriction sites were incorporated at the 5′ and 3′ end of theamplified NDM-1 and VIM-2 gene respectively. The NDM-1 and VIM-2 genewere then respectively inserted into plasmid pET-28a(+) (Novagen) inbetween the NdeI and EcoRI site so that an N-terminal His-tag wasengineered, facilitating protein purification by nickel affinitychromatography. The plasmid construct with the correct insert wassubsequently transformed into E. coli XL-1 Blue (Life Technologies) forcloning. The plasmid pET-28a(+) bearing the kanamycin-resistance genewas selected to avoid contamination of other β-lactamases with NDM-1 orVIM-2. All plasmid constructs of variants were transformed into E. coliXL-1 Blue (Life Technologies) for cloning. The DNA sequences of theconstructs were confirmed by DNA sequencing.

PCR primers for expressing NDM-1(29-270) and VIM-2_(21-266):

SEQ. ID NO. 1  NDM-1_29-270_for: GGGGGCATATGGGTGAAATCCGTCCGACSEQ. ID NO. 2 NDM-1_29-270_rev: GGGGGGAATTCTTAACGCAGTTTATCAGCCATSEQ. ID NO. 3 VIM-2_21-266_for: GGGGGCATATGAGCCCGCTGGCGTTTAGCGTGSEQ. ID NO. 4 VIM-2_21-266_rev: GGGGGGAATTCCACCACGCTGCGGTTGGTATG

6.3.2 Purification of Wild-Type NDM-1 and VIM-2

Wild-type NDM-1 or VIM-2 expression vector was subsequently transformedinto E. coli BL21(DE3) for protein expression. Fresh colony was pickedand inoculated in 1 L Luria-Broth medium supplied with 50 μg mLU⁻¹kanamycin at 37° C. until O.D. reaches 0.6. After the addition of 0.2 mMisopropyl β-D-1-thiogalactopyranoside and 0.2 mM ZnSO₄, the cells wereinduced to overexpress NDM-1 at 25° C. for 16-18 hrs. To purify thewild-type NDM-1 or VIM-2, cultured cells were harvested and lysed bysonication in a lysis buffer [20 mM HEPES/Na pH 7.0, 0.5 M NaCl and 1 mMphenylmethanesulfonyl fluoride (PMSF)]. The lysate was then centrifugedat 20,000 g for 30 mins to remove most of the insoluble cell debris. Thesupernatant was subsequently filtered using 0.45 μm filter (Sartorius)and loaded onto a Ni²⁺-NTA affinity chromatography column (GEHealthcare) at a rate of 2 mL min⁻¹. The column was washed using 5×column volume of washing buffer [20 mM HEPES, pH 7.0, 500 mM NaCl and 30mM imidazole] and the protein was eluted out using 4× column volume ofelution buffer [20 mM HEPES, pH 7.0, 500 mM NaCl and 300 mM imidazole].The N-terminal His-tag was cleaved by thrombin digestion at 25° C. for 3hrs using the cleavage buffer [20 mM HEPES/Na pH 7.0, 150 mM NaCl] andthe fusion His-tag was separated from NDM-1 by passing through theNi²⁺-NTA column again using washing buffer so that majority of the NDM-1protein was in the flow-through fraction. The proteins were furtherpurified in purifying buffer [20 mM HEPES, pH 7.0, 150 mM NaCl] usingHiLoad 16/60 Superdex 200 pg gel filtration column to a purity of >98%,judging from SDS-PAGE gel. The proteins were concentrated using AmiconUltra-15 Centrifugal Filter Devices (Millipore) and was separated intoaliquots in storage buffer [50 mM HEPES/Na pH 7.0] for storage at −20°C. The protein concentration was determined by using bicinchoninic acidprotein assay kit (Novagen) and the yield of the purified NDM-1 wasestimated to be 0.23 mg L⁻¹ of LB medium. Purified NDM-1 and VIM-2 weredetermined by ICP-MS to contain approximately 2 molar equivalents ofZn(II) (i.e. Zn₂-NDM-1) after the series of purification steps.

6.3.3 IC₅₀ Enzyme Inhibition Assay

Freshly prepared Zn₂-NDM-1 (50 nM) or Zn₂-VIM-2 were first incubatedwith various concentrations of Bi(III) compounds for 1 h at 25° C. Theassay was performed in a 1 cm quartz cuvette using the kinetic mode ofVarian Cary® 50 UV-visible spectrophotometer at 25° C. The final assaybuffer contains 50 mM HEPES/Na pH 7.0, 100 mM NaCl and 100 μM MER. Thedecrease in absorbance at 300 nm due to ring-opening of MER wasmonitored every second for a duration of 10 mins until the reaction wascompleted. The initial rate were extracted and calculated from eachreaction curves. Half maximum inhibitory concentrations (IC₅₀) of CBS toNDM-1 and VIM-2 were determined to be 5.83±0.53 μM and 44.45±3.94 μMrespectively.

6.3.4 Zn(II) Supplementation Assay

To investigate whether enzyme inhibition can be reversed, enzymeactivities of apo-NDM-1 and Bi-NDM-1 were compared with thesupplementation of Zn(II). Bi-NDM-1 (50 nM) was prepared bypre-incubation of apo-NDM-1 with CBS for 2 h at 25° C. and the bound Biwas verified using ICP-MS. The above protein solutions were mixed withZnSO₄ at concentration up to 2 molar equivalents to NDM-1 and 100 μMMER. The change in absorbance at 300 nm was monitored in a 1 cm quartzcuvette using Varian Cary® 50 UV-visible spectrophotometer at 25° C. forreaction rate calculations. Reaction rate of apo-NDM-1 with addition of2 molar equivalents of ZnSO₄ was normalized to be 1. It was found thatupon supplementation of Zn(II) to Bi-NDM-1 solution, the enzyme activitycan only be restored to 25%. In contrast, the enzyme activity can berecovered significantly upon the supplementation of Zn(II) to apo-NDM-1(FIG. 7). This undoubtedly suggests that Bi(III) inhibits NDM-1irreversibly and Zn(II) cannot reoccupy the active site to recover itsenzyme activity after Bi(III) binding. To confirm this, the metalcontents of both Zn(II) and Bi(III) were monitored byinductively-coupled plasma mass spectrometry (ICP-MS) as describedbelow.

6.3.5 Monitoring Zn(II) Displacement by Bi(III) Using ICP-MassSpectrometry (ICP-MS)

To monitor the replacement of Zn(II) by Bi(III), inductively-coupledplasma mass spectrometry (ICP-MS) was employed to accurately quantify²⁰⁹Bi and ⁶⁶Zn contents in various purified NDM-1 samples. PurifiedZn₂-NDM-1 was prepared as described previously and was dissolved intrace-metal-free ICP-MS buffer containing 50 mM HEPES, pH 7.0 throughcycles of buffer washing using an Amicon Ultra-15 Centrifugal FilterDevices with 3 kDa cut-off. Zn₂-NDM-1 (20 μM) was incubated with variousconcentrations of CBS at 25° C. for 5 hrs with mild shaking. The sampleswere subsequently dialyzed in ICP-MS buffer to remove unbound-metal ionsand were acidified with 10% (v/v) nitric acid for 1 h with gentleheating for releasing the bound metal ions from the protein for ICPanalysis. The samples were then mixed with 115In as an internal standardto reach a final concentration of 10 ppb in 1% HNO₃. After calibrationwith the isotope standards ²⁰⁹Bi, ⁶⁶Zn and ³⁴S from Sigma, the sampleswere subsequently injected into a quadrupole-based inductively coupledplasma mass spectrometer (Agilent 7500a, Agilent Technologies, CA, USA).²⁰⁹Bi, ⁶⁶Zn and ³⁴S isotopes abundances were determined simultaneouslyby integration of peak areas using the build-in software from theinstrument so that ³⁴S abundance serves as a way for quantifying proteinconcentration in each sample. It was found that 2.13 molar equivalentsof Zn(II) were replaced by 0.96 equivalent of Bi(III) as determined fromthe respective metal contents of the sample, providing direct evidencefor competitive binding of Bi(III) to the protein.

6.3.6 UV-Vis Spectroscopy

UV-vis spectra were collected on a Varian Cary 50 spectrophotometerusing a 1-cm quartz cuvette at 25° C. Freshly prepared apo-NDM-1 sample(50 μM) in 20 mM HEPES buffer containing 50 mM NaCl at pH 7.4 wasprepared and aliquots of bismuth solution were gradually titrated intothe sample. Binding of Bi(III) to apo-NDM-1 was monitored by measurementof the increase in absorption at approximately 340 nm due toligand-to-metal-charge transfer (LMCT) involving cysteine residue. SinceCys208 is the only cysteine residue in the protein, LMCT involvingcysteine residue provides solid evidence for Bi(III) binding to Cys208and subsequently leading to the dissociation of Zn(II) from NDM-1. Tofurther prove our hypothesis, X-ray crystallographic studies werecarried out as mentioned in Example 4 below.

6.4 Example 4: Crystallographic Studies of Bismuth Binding to NDM-16.4.1 Crystallization, Diffraction Data Collection and StructureDetermination

Crystals of native NDM-1 were obtained through hanging-drop vapordiffusion method. Briefly, NDM-1 protein (concentrated to 100 mg mL-1)was mixed with the precipitant containing 0.1 M Bis-Tris at pH 5.5, PEG3350 15% (w/v) and 20 mM L-proline in equal volume ratio. Under thiscondition, crystals in P1 space group with eight molecules in oneasymmetric unit could be obtained easily and they diffracted toresolution of 1.8 to 2.0 Å. After fishing up the P1 crystals, newcrystals in space group of P21 were grown by chance and they generallyhave greater diffraction limit to resolutions of 0.93˜1.0 Å. The P21crystals were used as seeds for later crystallization of NDM-1.Diamond-like or rectangular crystals appeared within a day after seedingand grew up to full size in three days to a week.

To obtain the bismuth bound crystals, zinc ions in the native crystalswere extracted and bismuth compounds were soaked into these crystals.Briefly, native crystals were crosslinked with 25% glutaraldehyde at 25°C. for 30 min and then soaked in chelating solution (0.1 M sodiumacetate pH 4.6, 25% PEG 3350, 20% glycerol, 10 mM EDTA) for anovernight. The crystals were washed three times in cryo-protectantsolution (0.1 M Bis-Tris pH 5.5, 25% PEG 3350, 20% glycerol). Soakingwas done in the soaking solution containing 0.1 M Bis-Tris (pH 5.5), 25%PEG 3350, 20% glycerol, 5 mM TCEP and 1 mM Bi(III) gluconate for 17 hrs.The crystals were then washed with the cryo-protectant solution for fourtimes and flash frozen into liquid nitrogen.

The diffraction data were collected at BL17U at the Shanghai SynchrotronRadiation Facility. Two data sets were collected at wavelengths of 0.92and 0.93 Å, which crossed the absorption edge of elemental bismuth.Excitation scan was performed for each crystals to confirm that zincions were completely extracted out and only bismuth was left in thecrystals. The diffraction data were processed with HKL2000. Molecularreplacement was performed using the program Phaser from CCP4 suit andthe ampicillin-bound NDM-1 (PDB code: 3Q6X) was used as a searchingmodel. Models were refined with Refmac and cycled with manual rebuildingin Coot. Anomalous data were used in refinement and the anomalous signalstrength was compared between the two data sets collected at wavelengthof 0.92 and 0.93 Å.

The anomalous peak for data collected at 0.92 Å was at least two-foldhigher than that of data collected at 0.93 Å. Together with theexcitation scan, we confirmed that the anomalous signal was contributedby bismuth. The occupancy of bismuth was refined according to thestrength of anomalous signal at early refinement and assessed byB-factor in later stages. TLS refinement was incorporated into laterrefinement processes. Solvents were added automatically in Coot and thenmanually inspected and modified. The final models were analyzed withMolProbity. Data collection and model refinement statistics aresummarized in Table 4.

TABLE 4 Data collection Bi(III)-gluconate Space group P21 Celldimensions a, b, c (Å) 41.60, 60.18, 41.80 α, β, γ(°) 90, 98.95, 90Resolution (Å)   50~1.55(1.61~1.55) Unique reflections 27612(2309) Completeness (%) 95.5(94.2) Redundancy 7.0(6.9) Wilson B-factor 12.79Rmerge 0.116(0.697) I/σI 18.1(3.1)  Refinement Resolution (Å) 50~1.55Rwork 0.1627(0.2194) Rfree 0.1935(0.2502) No. atoms 1951    Protein1737    Bi 1*  Water 206    Protein residues 228    Ramachandran plotFavored (%) 99   Outliers (%) 0   Average B-factors 19.62 Protein 17.90Water 33.77 Bia/Bib 14.94/18.42 Occupancy of Bia/Bib 0.55/0.10 R.m.s.deviations Bond lengths (Å)  1.37 Bond angles (Å)  0.010

6.5 Example 5: In Vitro Antimicrobial Activity Assessment

The anti-resistance activity of bismuth compounds was examined againstMBL producing bacteria strains. The method involved cell-based minimuminhibitory concentration (MIC) and minimum bactericidal concentration(MBC) assay, time-kill assay and in vitro cell infection assay.

6.5.1 Bacteria

The bacteria employed involved E. coli ATCC 25922, E. coli clinicalisolate (NDM-1⁺), E. coli (NDM-1⁻), K. pneumonia clinical isolate(NDM-1⁺), C. freundii clinical isolate (NDM-1⁺), E. coli clinicalisolate (IMP-4⁺), E. coli BL21 (VIM-2⁺) and Rosetta (NDM-1⁺). K.pneumonia clinical isolate (NDM-1⁺) and C. freundii clinical isolate(NDM-1⁺) were kindly given by Prof. Woo, Patrick Chiu Yat (LKS Facultyof Medicine, HKU). E. coli clinical isolate (IMP-4⁺) were kindly givenby Prof. Ho, Pak Leung (LKS Faculty of Medicine, HKU). E. coli (NDM-1⁻)was screened by 20^(th) generation serial passages of E. coli clinicalisolate (NDM-1⁺) in antibiotic-free medium. The missing of NDM-1 genewas confirmed by PCR check.

6.5.2 Microdilution MIC and MBC Assay

Both MIC and MBC assay were used to quantitatively evaluate theantimicrobial activity over the combination of MER and Bi(III) compoundsor pharmaceutically acceptable salts thereof. Fractional inhibitoryconcentration index (FICI) was used to mirror the synergism betweenthem.

MIC values of either antibiotics or Bi(III) compounds were determinedfirstly by standard broth micro-dilution method (Clinical and LaboratoryStandards Institute (CLSI) M100-S20, 2010). Briefly, bacteria cells werecultured in LB broth overnight at 37° C. at 250 rpm and the opticaldensity was measured at 600 nm (OD₆₀₀) using a microtiter plate reader.The bacteria density was adjusted to about 1×10⁶ CFU mL⁻¹ and checked byCFU counting on agar plates afterwards. Tested compounds were addedtriplicately into individual wells of flat-bottomed 96-well plates andperformed two-fold serial dilution, followed by addition of preparedbacterial inoculum. The plate was then incubated at 37° C. overnight.Lanes with no antibiotics or bismuth compounds served as positivecontrols and lanes with no bacteria added served as negative controls.The MIC was determined as the lowest concentration of compounds thatcould inhibit the growth of microorganism by both visual reading andOD₆₀₀ measurement. At the end of MIC assay, a 50 μL aliquot of each well(containing a specified antimicrobial concentration) for each isolatetested was applied to a LB agar plate and incubated at 37° C. in ambientair overnight. Resulting growth (or lack of growth) was examined afterovernight culturing and the lowest concentration that inhibits 99.9% ofthe original culture was taken as MBC.

For the test of drug combination, antibiotics and Bi(III) compounds wereco-administrated at concentrations up to 8 times higher than the MIC ofthe compounds tested alone. Other procedures and the check of MIC werekept strictly the same. The FICIs were determined based on the followingequation:FICI=FICA+FICB=C _(A)/MIC_(A) +C _(B)/MIC_(B)where MIC_(A) and MIC_(B) are the MIC values of compound A and B whenfunctioning alone, and C_(A) and C_(B) are the concentrations ofcompound A and B at the effective combinations. Synergism was defined asFICI≤0.5, indifference was defined as FICI>0.5 and <4, and antagonismwas defined as FICI≥4. All of the determinations were performed at leastin triplicate on different days.

Using the methods described above, exemplary Bi(III) compounds wereevaluated for their ability to kill or inhibit the growth of MBLproducing bacteria in the combination with MER. FIG. 10(a) shows theheat map of checkerboard MIC on the combination of MER and CBS againstNDM-1 producing E. coli. When used alone, MER had relatively high MICvalues, often greater than 16 μg mL⁻¹, which are far beyond theempirical susceptible level for Enterobacteriaceae (2 μg mL⁻¹). As theconcentration of CBS escalated, MIC of MER was gradually lowed to 2 μgmL⁻¹ and FIC index was calculated to be 0.250, indicative of synergisticeffect between them. In contrast, no inhibition was observed whenNDM-1-null E. coli stain was used (FIG. 10(b)). Such a synergy was alsofound between MER and other exemplary Bi(III) compounds against otherNDM-1-producing bacterial stains and other MBL-producing bacterialstains as summarized in Table 5, 6 and 7. Upon the combination withtested Bi(III) compounds, MICs of MER were substantially lowered,typically by 4˜32-folds against MBL-producing bacteria. Therepresentative results preliminarily indicate the synergism between theMER and tested Bi(III) compounds, which might contribute from theinhibition of MBL produced by the tested bacterial pathogens, consistentwith the previously described enzymatic study in Example 3.

TABLE 5 MER with Bi(III) compound at 32 μg mL⁻¹ Strain Compound MIC (μgmL⁻¹) MBC (μg mL⁻¹) FIC Index E. coli — 16 16 — (NDM-1⁺)  1 4 8 0.266  216 16 1  3 8 16 1  4 8 16 1  5 1 4 0.187  6 (BSS) 2 8 0.281  7 1 2 0.250 8 8 8 0.375  9 0.5 2 0.156 10 (CBS) 2 4 0.250 11 4 8 0.375 12 0.5 0.50.094 13 1 2 0.250 14 (BSG) 4 8 0.375 15 0.5 0.5 0.125 16 2 8 0.375 17 24 0.250 18 8 16 1 19 2 2 0.375 20 2 2 0.313 21 0.5 1 0.188 RBC 2 4 0.250Bi(NO3)3 2 4 0.266

TABLE 6 MER with Bi(III) compound at 32 μg mL⁻¹ MBC FIC Strain CompoundMIC (μg mL−1) (μg mL−1) Index K. pneumonia — 16 16 — (NDM-1⁺)  1 4 160.375 10 (CBS) 4 8 0.375 21 1 2 0.188 Bi(NO₃)₃ 1 1 0.125 C. feudii — 8 8— (NDM-1+) 10 (CBS) 0.5 1 0.125 21 0.5 0.5 0.188 Bi(NO₃)₃ 2 4 0.375

TABLE 7 MER with Bi(III) compound at 32 μg mL−1 FIC Strain Compound MIC(μg mL−1) MBC (μg mL−1) Index E. coli — 32 32 — (VIM-2⁺) 10 (CBS) 0.5 10.047 21 0.5 1 0.063 Bi(NO₃)₃ 1 2 0.063 E. coli — 8 16 — (IMP-4⁺) 10(CBS) 4 8 0.313 21 2 2 0.250 Bi(NO₃)₃ 4 8 0.313

TABLE 8 MER with CBS (mg mL⁻¹) at Mic multiple MER 32 64 128 2560.5 >1.31 × 10⁻⁷  >5.22 × 10⁻⁸  1.23 × 10⁻⁸ 9.84 × 10⁻¹⁰ 1.31 × 10⁻⁹1 >4.92 × 10⁻⁸  4.10 × 10⁻⁸ 1.15 × 10⁻⁹ — — 2 3.62 × 10⁻⁸ 3.28 × 10⁻⁹ —4 4.43 × 10⁻⁸ — 8 3.12 × 10⁻⁸ 16 — 32 64

The antibacterial activities of CBS with other β-lactam antibiotics wereshown in FIG. 11. Although none of the test combinations totallyinhibited the growth of bacteria, the growth rate was evidently loweredin Rosetta (DE3) (NDM-1⁺) bacterial cells by AMOX, ampicillin (AMP),nafcillin (NAF) and cefdinir (CEF) in the presence of bismuth complexes.AMOX, NAF and CEF becomes potent upon addition of compound (CPD) 21 withthe growth rates of the bacterial cells to be reduced by 77.9%, 60.5%and 61.1%, respectively, while CEF became extremely potent when CBS wasused in combination and could reduce the growth rate by 76%. This studydemonstrates the in vitro potency of the combination of bismuthcompounds with other antibiotics against NDM-1-producing bacteria.

6.5.3 Time Kill Assay

Time kill assay was used to further explore the synergy between MER andBi(III) compounds which were represented by CBS herein. In a typicalassay, the concentrations of the compounds used in this test representas follows: 16 μg mL⁻¹ MER, 32 μg mL−1 CBS. Bacteria were cultured forovernight and diluted 1:250 into LB broth at 37° C. for 3 hrs to reachlog phase. The initial bacterial concentration was adjusted to ˜10⁷ CFUmL⁻¹ according to standard curve. Tested compounds either alone or incombination was added to 20 mL of freshly prepared bacterial solution ina 50 ml tube, and incubate at 37° C. LB broth with no compounds servedas a positive control. Aliquots of 100 mL suspension were withdrawnafter different time intervals (0, 1, 2, 4, 6, 8, and 24 hrs). Bacterialconcentrations were estimated from colony counts by serial dilution inphosphate-buffered saline (PBS) and plating on LB agar. All assays weretriplicated and performed three times in different days. Data from threeindependent experiments were averaged and plotted as log₁₀ CFU mL⁻¹versus time (h) for each time point over 24 hrs as shown in FIG. 12.

In comparison to single components, bacteria density was significantlylowered by more than 1000-fold when exposed to the compound combinationof CBS and MER at the endpoint of the assay. According to NCCLS, thisindicates an evident synergism between CBS and MER and the bactericidaleffect over the compound combination was observed as well.

6.5.4 Resistance Study

Given the very notion of MIC, all susceptible bacterial cells will besuppressed or killed by a dose above it. However, bacterial populationis often large; an infection will likely contain first-step mutants withlowed susceptibility. Thus, the dose of compounds at MIC will adverselyamplify the population of those less-susceptible mutants.²⁶ Thus mutantprevention concentration (MPC), defined as a compound concentration thatsuppresses the growth of first-step resistant mutant in large quantityof susceptible bacterial population, together with mutant preventionindex (MPI=MPC/MIC), is introduced to estimate the mutant preventionability of exemplary compound combination.

For a typical test, E. coli cells (NDM⁺) at 1˜2×10¹⁰ CFU were spreadonto LB agar containing combinations of MER and CBS at identicalconcentrations. All the plates were incubated at 37° C. Upon incubationfor 48 hrs, up to 4 colonies were picked and re-cultured from any plateswith observable colonies, followed by the measurement of their MICvalues. Any MICs of MER that were higher than the original values (16 μgmL⁻¹) were determined as mutant colony. The concentration thatrestricted the growth of mutant colonies was determined as MPC.

A heat map visualized the degree of mutation frequency (FIG. 13), mutantcolonies were still observable even when a high dose (8×MIC) of MER wasapplied. In contrast, with the increase in CBS dose, the mutationfrequency declined significantly as shown in Table 7. The MPI of MER waslowered to 1 and no mutant colony was observable when ≥128 μg mL⁻¹ CBSwas used. This may contribute from multiple targets mechanism by usingbismuth compounds, which is believed as a typical feature ofmetalloagents. This unique character might play a vital role in endowingthe potency of Bi compounds to cope with resistance issue.

6.5.5 In Vitro Cell Infection Assay

To further evaluate the antimicrobial efficacy of CBS in combinationwith MER, we exploited the in vitro bacterial infection model inmammalian cell. Typically, MDCK cells were cultured in minimum essentialMedia (MEM) supplemented with fetal bovine serum (FBS, 10%) and grown at37° C. in the 5% CO₂ humidified atmosphere for three days. The MDCKcells were then washed three times with phosphate buffer saline (PBS)solution, liberated with trypsin-EDTA (0.25%) and re-suspended byculture medium. About 500 μL of re-suspended cells were seeded in24-well plates and incubated as described above for 48 hrs to ensureconfluency, resulting in about 1.0×10⁵ cells per well by typan blueassay. The freshly grown logarithmic E. coli (NDM-1⁺) bacterial cellswere washed with PBS for three times and re-suspended in MEM/10% FBS.The density was adjusted to 2×10⁷ CFU mL⁻¹. Then 500 μL of bacterialsuspension was added to each well and substituted for the previous MDCKculture medium. The plates were centrifuged at 800×g for 10 min and thenincubated at 37° C. in 5% CO₂ for 6 hrs executing the bacteria invasionat multiplicity of infection (MOI) of 200. After the infection, cellswere washed with PBS for three times to remove unbound bacteria andrefueled with culture medium.

For those cell-invaded bacteria experiments, the infected cells wereincubated in culture medium supplemented with 100 μg mL⁻¹ ciprofloxacinfor 1 h to remove extracellular bacteria. Then the treated cells werewashed vigorously with PBS for six times and replenished with culturemedium. To measure the initial bacteria density, cells were washed threetimes in PBS and fully lysed with of 1% Triton X-100 in PBS. The celllysates were serially diluted and plated on LB agar, and colonies wereenumerated by agar plating. For the cell-associated (adherent,internalized and transcytosed) infection, the cells were washedvigorously with PBS for washed six times in the absence of ciprofloxacintreatment. The cell-invaded bacteria herein are defined as thosebacteria that penetrate MDCK cells and the cell-associated bacteria aredefined as bacteria that penetrate, attach to or transcytose in MDCKcells. After the invasion of bacteria, appropriate concentrations oftested compounds were added to each well in 24-well plates. Cells in theabsence of the compounds served as a control. The plates were incubatedfor overnight at 37° C. Surviving bacteria were enumerated as describedpreviously. The assay was performed in triplicate, repeated three timesand the results were expressed as average ±SD.

FIG. 14 shows the CFU reduction of viable bacteria after the treatmentwith antimicrobial agent. Bacterial loads were at 10⁶ CFU level evenwhen MER at 2×MIC was used, which however, were dramatically dropped tonot greater than 10⁴ CFU level in the presence of CBS in thecell-associated model. For the cell-invaded model, the compoundcombination of MER (2×MIC) and CBS was still able to lower theintracellular bacterial density by 9.22 fold compared with mere MER. Thestudies above demonstrate the in vitro potency of the compoundcombination of CBS and MER against NDM-1-producing bacteria.

6.6 Example 6: In Vivo Antimicrobial Activity Assessments 6.6.1 MurinePeritonitis Infection

The enzyme- and cell-based study with which CBS served to suppress thefunction of MBLs and boost β-lactam antibiotic activity against MBLproducing bacteria demonstrates the potential that current compoundcombination would exert enhanced antimicrobial efficacy compared withβ-lactam antibiotic alone in vivo. To confirm this, a mouse peritonitismodel was established and then used for the examination of in vivocompound efficacy.

Briefly, an overnight culture of E. coli (NDM-1⁺) was performed 1:250dilution in 50 mL LB broth and re-grew to about OD of 0.3 in a 250-mLflask after 2.5 hrs shaking at 37° C., 250 rpm. The resulting bacterialpellets were collected and washed by PBS three times for further use.For bacterial infection, mice (female BALB/c strain, 6-8 weeks of age,18-22 g of weight) were induced intraperitoneally (i.p.) with a dose of10⁵ CFU bacteria in a 400 μL aliquot of PBS supplemented with 2% mucin.4 groups of mice were i.p. administered 4 hrs post-infection with a 100μL aliquot of PBS, MER, CBS and a combination of MER and CBS,respectively. Body weights and mice survival were monitored for endpointuntil day 4 post infection.

The representative results are shown in FIG. 15. The mice becameseverely diseased 4 hrs post-infection and none of them survived after20 hrs post-infection. Upon the co-administration with CBS, MER was ableto postpone the death of mice and raise the survival rate to 50%comparing with merely 25% by MER itself at the endpoint of experiment.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

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What is claimed is:
 1. A composition comprising: (a) a β-lactamantibiotic; and (b) a Bi(III) compound, or a pharmaceutically acceptablesalt thereof, wherein the Bi(III) compound is a complex comprisingBi(III) coordinated to a ligand selected from the group consisting of L1to L21:


2. The composition of claim 1 wherein the Bi(III) complex is a monomer,dimer or polymer.
 3. The composition of claim 2 wherein each of theBi(III) compounds or the pharmaceutically acceptable salt thereof has apH value in an aqueous solution of from about 3 to
 10. 4. Thecomposition of claim 1 wherein the β-lactam antibiotic and the Bi(III)compound, or a pharmaceutically acceptable salt thereof have a molarratio ranging from 1:16 to 16:1 by weight (w/w).